CN111032681A - Modified HSV gB protein and HSV vaccine comprising same - Google Patents

Modified HSV gB protein and HSV vaccine comprising same Download PDF

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CN111032681A
CN111032681A CN201880055586.8A CN201880055586A CN111032681A CN 111032681 A CN111032681 A CN 111032681A CN 201880055586 A CN201880055586 A CN 201880055586A CN 111032681 A CN111032681 A CN 111032681A
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森泰亮
西村知裕
清水裕之
松本美幸
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Km Biomedical Co Ltd
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Abstract

A modified HSV gB protein which is a modified protein of envelope glycoprotein B (gB) of Herpes Simplex Virus (HSV) and is formed by inactivating (deitopizing) at least 1 epitope in the epitopes (non-neutralizing epitopes) of induced non-neutralizing antibodies of domain IV and domain I of wild-type HSV gB.

Description

Modified HSV gB protein and HSV vaccine comprising same
Technical Field
The invention relates to a modified HSV gB protein and an HSV vaccine comprising the same.
Background
Herpes Simplex Virus (HSV) is a neurotropic pathogen that metastasizes to the sensory nerve after first infecting the mucosal epithelium, and remains latent for life in the trigeminal or sacral ganglia. Latent HSV is sometimes reactivated to cause various diseases (non-patent document 1).
HSV is known to exist in 2 serotypes (HSV-1, HSV-2), HSV-1 being the cause of orolabial and corneal herpes, HSV-2 being the cause of genital herpes. However, recently, due to the diversification of sexual behaviors, HSV-1 is frequently caused by genital herpes and HSV-2 is frequently caused by herpes labialis. The ratio of antibody-positive (infected) individuals in Japan is 60 to 80% in the case of HSV-1 and 10% in the case of HSV-2, and it is assumed that the potential requirement for a vaccine is 1000 million individuals even if only HSV-2 is used (non-patent document 2). In addition, the antibody-positive (infected) ratio in the U.S. is 57% in the case of HSV-1 and 20% in the case of HSV-2 (about 10% of them are marked genital herpes) (non-patent document 3).
It is known that 5 envelope glycoproteins (glycoprotens) are involved in the 2 stages of adsorption and invasion in the establishment of infection of cells by HSV. These 5 envelope glycoproteins are called envelope glycoprotein b (gb), envelope glycoprotein c (gc), envelope glycoprotein d (gd), envelope glycoprotein h (gh), and envelope glycoprotein l (gl), respectively (non-patent document 4).
First, it is believed that gB and gC bind to heparan sulfate on the cell surface, which is the trigger of the adsorption process (non-patent documents 5 and 6). This process is not essential when HSV invades cells, but is generally thought to be associated with more efficient invasion. Then, gB and gD bind to respective host cell receptors, and the viral envelope fuses with the host cell membrane, thereby initiating the invasion process.
As host cell receptors, gB receptors and gD receptors are known. As gB receptors, NM-IIA (non-patent documents 7 and 8) and MAG (non-patent document 9) have been identified. As gD receptors, bindin 1 (non-patent document 10), HVEM (non-patent document 11), and 3-O-sulfated heparan sulfate (non-patent document 12) have been identified. In addition, it is known that a gH/gL heterodimer interacts with gB and gD to play an important role in membrane fusion (non-patent document 13).
The structure of HSV-1gB was disrupted in 2006, and it was found that gB forms a trimer having 5 domains (non-patent document 14). In addition, the fact that gB has a structure similar to that of gG of VSV (Vesicular stomatitis virus) known as a membrane fusion protein supports that gB is a membrane fusion protein of HSV. In addition, gB is also highly conserved among other herpesviruses, and its function is considered to be identical among herpesviruses.
Pathogens causing infections are broadly classified into: group I pathogens that achieve sufficient efficacy with existing vaccines and group II pathogens that do not achieve sufficient protective immunity with existing vaccines, with a history of pathogen infection. It has been pointed out that the reason for the difficulty in defense of group II group pathogens of Class is the ingenious immune escape mechanism they possess (non-patent document 15). HSV belongs to the Class II group of pathogens, which we believe is due to: HSV has an immune escape mechanism and can skillfully escape the immune response of a host. Attempts have been made to develop HSV vaccines by using live attenuated vaccines and inactivated adjuvanted vaccines, but in any case, T-cell and B-cell immune responses are insufficient, and there is no difference from the level of insufficient immune responses obtained after natural infection.
Documents of the prior art
Non-patent document
Non-patent document 1, Roizman, B, et al, Herpes simple viruses, p.2501-2602, InD.M.Knipe and P.M.Howley (ed.), "Fields Virology", 5th ed.Lippincott Williams & Wilkins, Philadelphia, P.A.2007
Non-patent document 2, Hashido M1, etc., epidemic study of diseases simplexvirus type 1and 2infection in Japan based on type-specific serological analysis, epidemic infection in 1998 Mar; 120(2):179-86
Non-patent document 3, precision Resources; emerging Vaccines 2008
Nonpatent document 4: ウイルス 2010 No. 60, No. 2, pp.187-196, volume 2010
Non-patent document 5: Herold, B.C. et al, Glycoprotein C-independent binding of proteins from cells to cells requirement cell surface carbohydrate B.J Gen Virus 199475 (Pt 6):1211-22
Non-patent document 6: Herold, B.C. et al, Glycoprotein C of skin simple virus type1 plate a primary roll in the administration of virus to cells and administration.J. roll 199165: 1090-8
Non-patent document 7, Arii, J.et al, Non-muscle myostatin IIA a functional regenerative for drugs simple virus-1.Nature 2010467: 859-62
Non-patent document 8, Satoh, T, et al, PILRalpha is a peptides simple virus-1 entryyceteceptor that is associated with a virus B.cell 2008132: 935-44
Non-patent document 9, Suenaga, T.et al, Myelin-associated glycoprotein media fusion and entry of neuropic heat drugs, Proc Natl Acad Sci U SA 2010107: 866-71
Non-patent document 10 Geright, R.J., et al, Entry of alphaviruses meditated branched protein 1and poliovirus receptor science 1998280:1618-20
Non-patent document 11, Montgomery, R.I., and the like, and Herpes simple virus-1entry by a novel member of the TNF/NGF receiver family.cell (1996)87:427-36
Non-patent document 12, Shukla, D, et al, A novel roll for 3-O-sulfonated heparin in peptides simple virus 1entry. cell 199999: 13-22
Non-patent document 13 Eisenberg RJ et al, and Herpes viruses fusion and entry a stores with machines, viruses 20124: 800-83210.3390/v 4050800
Non-patent document 14 SCIENCE 2006313, 14,217-220
Non-patent document 15 Vaccine 26(2008)6189-
Non-patent document 16 The Journal of Immunology,1997, 159279-289.
Disclosure of Invention
Problems to be solved by the invention
As described above, antiviral drugs such as acyclovir are used in the treatment of HSV. However, these antiviral agents cannot completely eliminate the virus, and the virus is reactivated when the administration is stopped. Therefore, development of a prophylactic vaccine for protection against HSV infection or a therapeutic vaccine for alleviating relapsing symptoms is desired, but no effective vaccine exists at present, and there is a high demand for the vaccine.
The present invention provides a modified HSV gB protein which, upon immune induction, can induce an antibody containing a high proportion of a neutralizing antibody showing high neutralizing activity against HSV gB, as compared with wild-type HSV gB, and can be used for prevention and/or treatment of HSV infection, and a vaccine comprising the same.
Means for solving the problems
For the gB protein, which is known to be one of the major defense antigens of HSV, the inventors have conducted an exhaustive B cell epitope analysis in an attempt to classify it into an epitope that is beneficial in exhibiting defensive activity (neutralizing epitope) and an epitope that is not beneficial or harmful in exhibiting defensive activity (non-neutralizing epitope). Then, the useless or harmful epitope is subjected to de-epitopation, and the beneficial epitope is immunologically highlighted, so that the modified HSV gB protein and the vaccine containing the modified HSV gB protein, which can enhance the induction capability and the anti-infection capability of the neutralizing antibody, are completed.
That is, the present invention relates to the following inventions.
(1) A modified HSV gB protein which is a modified protein of the envelope glycoprotein b (gB) of Herpes Simplex Virus (HSV) (modified HSV gB protein) which is a modified HSV gB protein modified as follows: at least 1 of the non-neutralizing antibody-inducing epitopes (non-neutralizing epitopes) present in domain IV and domain I of wild-type HSV gB was not rendered to function as a volatile epitope.
(2) The modified HSV gB protein according to (1), wherein the non-neutralizing epitope is an epitope comprising at least 1 amino acid residue present in a region which is located at a distance of 1.5nm or less from an amino acid residue corresponding to arginine residue 567 (R567), arginine residue 602 (R602), serine residue 631 (S631), or aspartic acid residue 199 (D199) in the amino acid sequence described in SEQ ID NO. 1, on the surface of the crystal structure of the extracellular domain of wild-type HSV gB.
(3) The modified HSV gB protein according to (1) or (2), wherein the non-neutralizing epitope is an epitope comprising the amino acid residue corresponding to R567, R602, S631 or D199 in the amino acid sequence shown in SEQ ID NO. 1.
(4) The modified HSV gB protein according to any one of (1) to (3), wherein the modification comprises a modification by substitution of an amino acid residue and/or deletion of an amino acid residue.
(5) The modified HSV gB protein according to (4), wherein the modification comprises a modification by introducing a sugar chain by substitution or deletion with an amino acid residue.
(6) The modified HSV gB protein according to any one of (1) to (5), wherein the modification comprises a modification for sugar chain introduction at a position of at least 1 amino acid residue selected from the group consisting of amino acid residues corresponding to D199, R567, R602, and S631 in the amino acid sequence shown in SEQ ID NO. 1.
(7) The modified HSV gB protein according to any one of (1) to (6), wherein the modification comprises a modification for sugar chain introduction at a position of at least 2 amino acid residues selected from the group consisting of the amino acid residues corresponding to D199, R567, R602, and S631 in the amino acid sequence shown in SEQ ID NO. 1.
(8) The modified HSV gB protein according to (7), wherein the modification comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residues R567 and S631 in the amino acid sequence of SEQ ID NO. 1.
(9) The modified HSV gB protein according to (8), wherein the modification comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residues R567 and S631 in the amino acid sequence of SEQ ID NO. 1.
(10) The modified HSV gB protein according to (7), wherein the modification comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residues D199, R567 and S631 in the amino acid sequence of SEQ ID NO. 1.
(11) The modified HSV gB protein according to any one of (6) to (10), wherein the modification comprises a modification for sugar chain introduction at a position corresponding to the amino acid residue of R602 in the amino acid sequence shown in SEQ ID NO. 1.
(12) The modified HSV gB protein according to (11), wherein the sugar chain has been introduced by replacing the amino acid residues R602N, D603A and A604T in the amino acid sequence of SEQ ID NO. 1.
(13) The modified HSV gB protein according to any one of (5) to (12), wherein the modification further comprises a modification for sugar chain introduction at a position corresponding to the amino acid residue D199 in the amino acid sequence shown in SEQ ID NO. 1.
(14) The modified HSV gB protein according to (13), wherein the sugar chain is introduced by replacing the amino acid residues D199N, D200A and H201T in the amino acid sequence of SEQ ID NO. 1.
(15) The modified HSV gB protein according to any one of (4) to (14), wherein the modification further comprises substitution of the amino acid residue corresponding to arginine (R613) at position 613 in the amino acid sequence of SEQ ID NO. 1 with an alanine residue.
(16) An HSV vaccine comprising the modified HSV gB protein of any one of (1) to (15).
(17) A modified HSV gB protein which is a modified protein of the envelope glycoprotein B (gB) of Herpes Simplex Virus (HSV), wherein at least 1 amino acid residue present in a region which is located at the surface of the crystal structure of the extracellular domain of wild-type HSV gB and which has a distance of 1.5nm or less from the amino acid residue corresponding to arginine residue 567 (R567), arginine residue 602 (R602), serine residue 631, or aspartic acid residue 199 (D199) in the amino acid sequence shown in SEQ ID NO. 1, is substituted for or deleted from the modified HSV gB protein.
(18) The modified HSV gB protein according to (17), wherein the modification comprises a modification for sugar chain introduction at a position of at least 1 amino acid residue selected from the group consisting of amino acid residues corresponding to D199, R567, R602, and S631 in the amino acid sequence described in SEQ ID NO. 1.
(19) The modified HSV gB protein of (17) or (18), wherein the modification comprises a modification for sugar chain introduction at a position corresponding to the amino acid residue D199 in the amino acid sequence of SEQ ID NO. 1.
(20) The modified HSV gB protein according to any one of (17) to (19), wherein the modification comprises a substitution of the amino acid residue corresponding to arginine (R613) at position 613 in the amino acid sequence of SEQ ID NO. 1 with an alanine residue.
(21) The modified HSV gB protein according to any one of (17) to (20), wherein the modification comprises a modification for sugar chain introduction at a position corresponding to the amino acid residue R567 in the amino acid sequence shown in SEQ ID NO. 1.
(22) The modified HSV gB protein according to any one of (17) to (21), wherein the modification comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residue S631 in the amino acid sequence of SEQ ID NO. 1.
(23) An HSV vaccine comprising the modified HSV gB protein of any one of (18) to (22).
ADVANTAGEOUS EFFECTS OF INVENTION
When the modified HSV gB protein of the present invention and a vaccine comprising the same are used for immune induction, relatively more neutralizing antibodies having high neutralizing activity may be contained in serum than when the modified HSV gB protein is used for immune induction. Namely, the modified HSV gB protein and the vaccine containing the same can induce immune refocusing and bring a strong defense effect on HSV. Therefore, a high prophylactic and therapeutic effect on HSV infection can be expected.
Drawings
FIG. 1 is a graph showing the results of SDS-PAGE and Western blotting using gB and its protease-cleaved fragment of example 2.
FIG. 2 is a graph showing the correlation between the anti-gB antibodies obtained by competitive ELISA in example 2.
FIG. 3 is a MOE diagram showing the identification result of the epitope of the gB antibody obtained by alanine scanning in example 3.
Fig. 4 is a diagram showing a schematic of the design strategy for modified gB proteins of example 5.
Fig. 5 is a diagram showing a simplified diagram of the crystal structure of the design strategy for a modified gB protein of example 5.
Fig. 6 is a graph showing the results of the mouse immunogenicity test of bcev19 of example 5.
Fig. 7 is a graph showing the results of the mouse immunogenicity test of bcev50 of example 5.
Fig. 8 is a graph showing the results of the survival rate of the mouse anti-infection test of bcev19 of example 5.
Fig. 9 is a graph showing the results of symptom scores of the mouse anti-infection test of bcev19 of example 5.
Fig. 10 is a graph showing the results of the survival rate of the mouse anti-infection test of bcev50 of example 5.
Fig. 11 is a graph showing the results of symptom scores of the mouse anti-infection test of bcev50 of example 5.
Fig. 12 is a graph showing the results of an analysis of the immune refocusing of bcev19 of example 5.
Fig. 13 is a graph showing the results of an analysis of the immune refocusing of bcev50 of example 5.
FIG. 14 shows the results of the analysis of the properties of bcev19, bcev19 ', bcev50 and bcev 50' by gel filtration chromatography in example 5.
Fig. 15 is a graph showing the results of comparing bcev19 with bcev 19' of the mouse immunogenicity test of example 5.
Fig. 16 is a graph showing the results of comparing bcev50 with bcev 50' of the mouse immunogenicity test of example 5.
FIG. 17 is a diagram showing the results of comparison of a multiple alignment of an HSV-1-derived gB amino acid sequence (SEQ ID NO: 2) and an HSV-2-derived gB amino acid sequence (SEQ ID NO: 3), in which italics indicate a leader sequence and underlines indicate the amino acid residues (I383-R388) at position 383 and 388 of the domain II of the HSV-1-derived gB and the amino acid residues (I386-R391) at position 386 and 391 of the domain II of the HSV-2-derived gB.
Detailed Description
The mode for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.
The modified HSV gB protein is a modified protein of envelope glycoprotein B (gB) of Herpes Simplex Virus (HSV), and is modified as follows: at least 1 epitope among the non-neutralizing antibody-inducing epitopes (non-neutralizing epitopes) present in domain IV and domain I of wild-type gB is made not to function as an epitope.
The present invention is based on the hypothesis proposed by the inventors that a "bait region" is present in the HSV gB antigen. The "Decoy region" is derived from "Decoy" in english and is considered one of the immune escape mechanisms by which pathogens escape the host's immune response. The "bait region" is an antigen region of an antibody that induces no neutralizing antibody activity or an antibody that has low neutralizing antibody activity, and the mechanism is generally considered as follows: the pathogen evades the host immune response by virtue of the fraudulent imprinting (also called "immunological deviation") resulting in no or low production of neutralizing antibodies.
To date, the presence of a bait zone in HSV has not been identified, nor has the concept of a bait zone. The present inventors performed detailed epitope mapping analysis on an anti-gB monoclonal antibody obtained by exhaustive search of anti-HSV gB antibodies using a human antibody library. The results show for the first time that domain IV and domain I of HSV gB are decoy regions where useless or harmful epitopes are concentrated. And immunologically highlighting the beneficial epitope by deitoping the non-neutralizing epitope in the bait region, thereby obtaining a modified HSV gB protein capable of inducing antibodies with high neutralizing activity.
"wild-type HSV gB" means: the full-length HSV-1-derived envelope glycoprotein b (gB) having the amino acid sequence shown in seq id No. 2 or HSV-2-derived gB having the amino acid sequence shown in seq id No.3 were aligned in multiple ways, and as a result of comparison, the sequence homology was about 87% (fig. 17). The three-dimensional structure of gB was also analyzed and was known to consist of an intracellular domain, a transmembrane domain and an extracellular domain. For example, Science 313: 217-220(2006) and J.Virol.84: 12924-12933(2010) reports the crystal structure of HSV-1 derived gB. On the other hand, the crystal structure of HSV-2-derived gB has not been reported, but it can be analyzed in the same manner as the above-mentioned method for crystallizing HSV-1-derived gB. By "extracellular domain of wild-type HSV gB" is meant the soluble, antigenic, extracellular region of wild-type HSV gB. An example of the extracellular domain of wild-type HSV gB is the 333-strain wild-type gB extracellular domain 1-705 of HSV-2 having the amino acid sequence shown in SEQ ID NO. 1.
In the crystal structure of gB, domain IV and domain I located at the upper part (top) and lower part (bottom), respectively, are more "conspicuous" and have high antigen presentation properties than domain II located at the Middle part (Middle), and according to the investigation of the present inventors, the proportion of antibodies against domain IV and domain I is actually high in the antibodies in blood, and the proportion of antibodies against domain II is low. On the other hand, according to the studies of the present inventors, it was also known that only an epitope inducing a neutralizing antibody (referred to as "neutralizing epitope" in the present specification) was present in domain II of wild-type HSV gB, whereas both an epitope inducing a neutralizing antibody and an epitope inducing a non-neutralizing antibody (referred to as "non-neutralizing epitope" in the present specification) were present in domain IV and domain I of wild-type HSV gB. Non-neutralizing antibodies, although binding to an antigen (i.e., a virus), do not inhibit the activity of the virus, and therefore it is important to induce the production of neutralizing antibodies, rather than non-neutralizing antibodies, in the manufacture of vaccines.
The modified HSV gB protein of the present invention can induce antibodies with high neutralizing activity by "inconspicuous" by de-epitopic "domain IV and domain I, which are less beneficial in the production of neutralizing antibodies, thereby" visualizing "domain II with a neutralizing epitope.
"modified HSV gB protein" (also referred to as "modified protein of HSV gB" or "modification") refers to: proteins in which at least one amino acid residue or a continuous amino acid residue region is substituted, deleted or added for wild-type HSV gB include proteins modified with proteins not present in wild-type, such as proteins having a sugar chain introduced by substitution or deletion of an amino acid residue. Compared with wild type protein, the modified HSV gB protein has high induction activity of a neutralizing antibody.
By "neutralizing antibody inducing activity" is meant: the ability to induce neutralizing antibodies against an antigen protein can be evaluated by the neutralizing antibody titer (neutralizing antibody titer) in immune serum obtained by inoculating an animal to be tested with an antigen protein. By "neutralizing antibody" is meant: the antibody that renders the virus particle non-infectious can be evaluated for the intensity of the neutralizing activity by, for example, the concentration of the antibody required to reduce the plaque number of the test virus by 50% (NT 50).
"Deepitopic" means: the modification was performed in such a manner that the site in wild-type HSV gB that contributes to antibody production as an epitope does not function as an epitope. Examples of the method of de-epitopic formation include: a method of substituting an amino acid residue at an epitope site with another amino acid residue, a method of deleting (deleting) an amino acid residue at an epitope site, a method of introducing a sugar chain by substitution or deletion of an amino acid residue at an epitope site, and the like. As a method for de-epitopic formation, a method of introducing a sugar chain, particularly a method of introducing an N-type sugar chain (N-glycoside-bonded sugar chain), is preferable. This is effective because not only the part to which the sugar chain is introduced but also the surrounding epitopes are simultaneously masked due to its large volume. Considering the size ratio of the protein such as an antibody or receptor interacting with gB, it is estimated that the possibility of utilizing point-to-point interaction in which a bond is formed in an extremely narrow range of about a few amino acids is low. It is considered that when gB binds to a receptor, an interaction network is formed between the surface and the pass plane, such that a wide range of amino acids synergistically form a bond. It is considered that sugar chain introduction is an effective epitope-removing method that widely blocks peripheral residues by its large volume and suppresses approach (access) of a binding partner. In addition, it has been reported that the sugar chain is less likely to induce an anti-sugar chain antibody, and it is considered that the possibility of the generation of new immunogenicity due to modification is suppressed to a low level.
As an example of the epitope (non-neutralizing epitope) for inducing a non-neutralizing antibody present in the domain IV and the domain I of wild-type gB, an epitope including at least 1 amino acid residue present in a region at a distance of 1.5nm or less from the amino acid residue corresponding to the arginine residue at position 567 (R567), the arginine residue at position 602 (R602), the serine residue at position 631 (S631), or the aspartic acid residue at position 199 (D199) in the amino acid sequence described in sequence No. 1, more preferably a region at a distance of 1nm or less from the amino acid residue, on the surface of the crystal structure of the extracellular domain of wild-type HSV gB is exemplified. Here, the "distance from … … amino acid residue" refers to a straight-line distance from the above amino acid residue, regardless of the surface shape of the crystal structure of the extracellular domain of wild-type HSV gB. Non-neutralizing epitopes induce the production of useless or harmful antibodies, and are useless for the production of neutralizing antigens, so that the de-epitopic formation of these can reduce the production of useless or harmful antibodies, and additionally highlight the beneficial epitopes, so that the production of neutralizing antibodies can be increased. The crystallization method is not particularly limited, and j.virol.84: 12924-12933 (2010). For example, 15% PEG4000-0.3M NaCl-0.1M sodium citrate (pH5.5) can be used to grow crystals of gB.
Examples of the non-neutralizing epitope include an epitope including an amino acid residue corresponding to arginine residue 567 (R567), arginine residue 602 (R602), serine residue 631, or aspartic acid residue 199 (D199) in the amino acid sequence of seq id No. 1. The inventors have demonstrated that these epitopes are non-neutralizing epitopes and thus de-epitopic of these epitopes can reduce the production of antibodies that are not beneficial or detrimental in exhibiting defensive activity. In addition, de-epitopic modification of these epitopes can increase the rate of production of neutralizing antibodies by highlighting beneficial epitopes.
Modifications for epitope removal include modifications by substitution of amino acid residues, deletion of amino acid residues, and/or introduction of sugar chains based on substitution or deletion of amino acid residues.
The modification preferably includes a modification for introducing a sugar chain at a position of at least 1, preferably at least 2 amino acid residues selected from the group consisting of amino acid residues corresponding to aspartic acid residue 199 (D199), arginine residue 567 (R567), arginine residue 602 (R602), and serine residue 631 in the amino acid sequence of seq id No. 1. It is preferable to include modifications for sugar chain introduction at positions corresponding to the amino acid residues of R567 and S631 in the amino acid sequence of SEQ ID NO. 1, respectively, by amino acid residue substitution of R567N, P568S, G569S, S631N, H632T and Q633T in the amino acid sequence of SEQ ID NO. 1. Further, it is preferable to include a modification for introducing a sugar chain at a position corresponding to each of the amino acid residues D199, R567 and S631 in the amino acid sequence of SEQ ID NO. 1. The modification preferably further comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residue of R602 in the amino acid sequence of SEQ ID NO. 1, preferably by substitution of the amino acid residues of R602N, D603A and A604T in the amino acid sequence of SEQ ID NO. 1. The modification preferably further comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residue of D199 in the amino acid sequence of SEQ ID NO. 1, preferably by substitution of the amino acid residues of D199N, D200A and H201T in the amino acid sequence of SEQ ID NO. 1.
The method for introducing a sugar chain is not particularly limited, and for example, in the case of introducing an N-type sugar chain, a primer is designed using cDNA (GenBank: M15118.1, SEQ ID NO: 4) of the wild-type gB protein as a template so that 3 consecutive amino acid sequences of a target site to be introduced into the N-type sugar chain are N-X-S/T (X is an arbitrary amino acid other than proline), and a mutation is introduced by PCR. Examples of the mutation for introducing a sugar chain include the following mutations in the amino acid sequence of SEQ ID NO. 1: (D199N, D200A, H201T), (R567N, P568S, G569S), (S631N, H632A, Q633T) and (S631N, H632T, Q633T). The gB modified form can be obtained by cloning and expressing a nucleic acid sequence of the target modified gB protein and, if necessary, a nucleic acid sequence to which a tag such as 6 × His is linked into an appropriate vector. Then, an N-type sugar chain is added to asparagine at the target site of the gB-modified body by a conventional method.
Modification of a non-neutralizing epitope may further comprise replacement of the charged amino acid residue as the epitope with an uncharacterized amino acid residue, such as an alanine residue. This method has an advantage that epitope can be removed accurately, unlike the introduction of an N-type sugar chain. For example, the alanine substitution preferably includes substitution of an amino acid residue corresponding to arginine (R613) at position 613 in the amino acid sequence of seq id No. 1 with alanine.
Preferred as the modified HSV gB protein is a gB protein in which a sugar chain is introduced into each of the amino acid residues corresponding to D199, R567 and S631 in the amino acid sequence of seq id No. 1, and the amino acid residue corresponding to arginine (R613) at position 613 in the amino acid sequence of seq id No. 1 is substituted with alanine. As another example of the modified HSV gB protein, a gB protein in which a sugar chain is introduced into each of the amino acid residues corresponding to D199, R567, R602, and S631 in the amino acid sequence of seq id No. 1, and the amino acid residue corresponding to arginine (R613) at position 613 in the amino acid sequence of seq id No. 1 is substituted with alanine is more preferable.
The modified HSV gB protein of the invention can be prepared by a genetic engineering method. The production method is not particularly limited, and for example, the following can be obtained: a primer for introducing a desired mutation is designed using cDNA of a wild-type gB protein as a template, a nucleic acid into which a mutation has been introduced is obtained by PCR, functionally linked to an expression promoter, and optionally linked to a tag, and introduced into an appropriate expression vector to express the nucleic acid. In addition, in the case of a sugar chain-introduced modified product, it can be obtained as described above.
The prepared modified HSV gB protein can be purified as required. The purification method is not particularly limited, and purification by an affinity column or the like is exemplified.
HSV infection includes HSV-1 and HSV-2 caused by infection, such as herpes labialis, corneal herpes, genital herpes, systemic neonatal herpes, and HSV caused by stomatitis, dermatosis, encephalitis, myelination and myelitis.
The HSV vaccine of the present invention comprises the modified HSV gB protein of the present invention.
The dosage form of the HSV vaccine of the present embodiment may be, for example, a liquid, a powder (lyophilized powder, dried powder), a capsule, a tablet, or a frozen state.
The HSV vaccine of the present embodiment may comprise a pharmaceutically acceptable carrier. The carrier may be any one commonly used for the production of vaccines without limitation, and specific examples thereof include saline, buffered saline, dextran, water, glycerol, isotonic aqueous buffer, and combinations thereof. The vaccine may further contain an emulsifier, a preservative (e.g., thimerosal), an isotonicizing agent, a pH adjuster, and the like as appropriate.
To further improve the immunogenicity of the HSV vaccine of the present embodiments, an adjuvant may be further included. Examples of adjuvants include aluminum adjuvants, squalene-containing oil-in-water emulsion adjuvants (such AS AS03 and MF 59), Toll-like receptor ligands such AS CpG and 3-O-deacylated-4' -monophosphoryl lipid A (MPL), saponin adjuvants, polymer adjuvants such AS poly-gamma-glutamic acid, polysaccharides such AS chitosan and inulin.
The HSV vaccine of the present embodiment can be obtained by mixing the modified HSV gB protein of the present invention with a carrier, an adjuvant, and the like as needed. Adjuvants may be of the type that are mixed on site.
The route of administration of the HSV vaccine of the present embodiment may be, for example, transdermal administration, sublingual administration, eye drop administration, intradermal administration, intramuscular administration, oral administration, intestinal administration, nasal administration, intravenous administration, subcutaneous administration, intraperitoneal administration, or inhalation administration from the mouth to the lung.
The HSV vaccine of the present embodiment may be administered, for example, by a syringe, a transdermal patch, a microneedle, a transferable sustained release device, a syringe with a microneedle, a needleless device, or a spray.
Examples
The present invention will be described in more detail below based on examples. However, the present invention is not limited to the following examples.
Example 1 isolation of anti-gB antibodies
The cDNA (SEQ ID NO: 4) of the gB extracellular domain 1-705aa (gB1-705) from strain 333 of HSV-2 was cloned into pCAGGS 1-dhfr-neo. Designed to add a streptavidin Tag II (strep Tag II) at the C-terminus of gB. For expression, FreeStyle293 or Expi293 expression systems (Life Technology) were used. The expression plasmid was transfected into cells, and the culture supernatant was recovered at 4 to 6 days. To remove biotin contained in the medium, the culture supernatant containing gB was concentrated with a UF membrane. The concentrated culture supernatant was purified by a StrepTactin column to obtain purified gB 2. The gene encoding amino acids 370 to 457aa of gB was amplified by PCR and cloned into pet43.1b (+) to construct a gB370-457 expression plasmid. Nus-Tag gene and His-Tag gene were added to the plasmid for easy purification. The plasmid was transformed into Rosetta2 (Novagen). After culturing in LB medium at 37 ℃ until the logarithmic growth phase, expression was induced with 1mM IPTG and cultured overnight at 25 ℃. Soluble proteins were extracted using a BugBuster mix. The resulting protein was purified using Ni NTA Agarose (QIAGEN).
44 scFv clones reactive to gB of HSV-2 were isolated by screening single-chain antibody phage (scFv-phase) display libraries prepared from human B-cell-derived mRNAs derived from tonsils and spleens using human VH and VL cDNAs. The DNA nucleotide sequences of the isolated VH chain and VL chain genes of the scFv gene were determined using Big Dye terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Any clone has an independent sequence, and thus variation in its epitope is expected. Multiple scFv clones were obtained outside of these 44 clones, but most of them had sequences similar to D1 or D2 (data not shown). D1 and D2 are VH5 family members, and are rare as antibody populations in organisms.
Example 2 Classification of anti-gB antibodies
< preparation of scFv-hFc antibody fragment >
The variable region of the isolated scFv gene was ligated with the human Fc gene, and cloned into pCAG vector to construct scFv-hFc expression plasmid. For expression, FreeStyle293 or Expi293 expression systems were used. The expression plasmid was transfected into cells, and the culture supernatant was recovered at 4 to 6 days. For the culture supernatant, purification of scFv-hFc was performed using Ab Rapid PuRe 10 (ProteNova). In addition, Escherichia coli TG1 strain having a phagemid vector in which each scFv gene was cloned was cultured in 2 XYTCG medium (37 ℃ C.), M13K07 helper phage was infected with moi ═ 20, and then phage expression was performed overnight in 2 XYTCK medium (25 ℃ C.). The obtained single-chain antibody phage was concentrated using 20% -PEG-2.5M NaCl.
The binding activity of the single-chain antibody phage to scFv-hFc was evaluated by ELISA. The recombinant gB was immobilized by diluting gB1-705 to 1. mu.g/mL with PBS, adding 50. mu.L to Maxi Sorp plate (Nunc), and incubating at 4 ℃ overnight or at room temperature for 1-2 hours. After immobilization, the plate was washed with PBS, and 100. mu.L of the resulting single-chain antibody phage and IgG was added to the well of the plate, followed by incubation at 37 ℃. After 1 hour PBST washing was performed, and 100. mu.L of detection antibody anti-M13/HRP (GEHealthcare) or anti-hFc/HRP (BIO COSMO) was added to the wells of the plate and incubated at 37 ℃. After 1 hour, the plate was washed with PBST, and 100. mu.L of TMB was added to the wells of the plate to develop color. After 30 minutes the reaction was stopped with 1N sulfuric acid and the absorbance (O.D.450nm/650nm) was measured with a microplate reader (molecular devices) (data not shown).
< Classification of anti-gB antibodies Using Western blotting Using gB and protease-cleaved fragment thereof >
In order to classify the obtained 44 anti-gB antibody clones, Western blotting was performed using gB fragments obtained by digesting gB1-705 with trypsin or chymotrypsin, gB1-705, and cleavage mutants thereof as materials.
500ng of denatured or undenatured gB1-705 was loaded onto 8-16% SDS-PAGE for electrophoresis. The denatured gB1-705 was obtained by adding 2% 2-mercaptoethanol to gB1-705 and boiling at 96 ℃ for 5 minutes. The non-denatured gB1-705 was not subjected to these manipulations but directly loaded. The gB fragment obtained by digestion with trypsin or chymotrypsin was obtained as follows. 500ng of gB1-705 and 1ng, 5ng or 1000ng of trypsin or 1ng of chymotrypsin were added to 0.5M Tris-HCl (pH8.0), and the mixture was allowed to stand at 37 ℃ for 1 hour or 3 hours for enzyme treatment, and SDS-PAGE loading buffer supplemented with 2% 2-mercaptoethanol (Wako) was added thereto and boiled at 96 ℃ for 5 minutes to terminate the reaction. Each subject was subjected to 8-16% SDS-PAGE, and the gel was transferred to a nitrocellulose Membrane (MILLIPORE) and blocked with 2% skim milk/PBS-T. After washing with PBS-T, each scFv-hFc was reacted in 1. mu.g/mL of 2% skim milk-PBS-T at room temperature for 30 minutes. After washing again, anti-hFc/HRP was reacted in 2% skim milk-PBS-T and developed with Immobilon WesternDetection Regent (Millipore). Silver staining was performed in the band detection of SDS-PAGE.
The results are shown in FIG. 1. The subjects in the lanes of SDS-PAGE and Western blot in FIG. 1 are as follows. In SDS-PAGE, lane 1: non-reducing undenatured gB1-705, lane 2: boiling only gB1-705, lane 3: boiled and denaturant denatured gB1-705, lane 4: gB370-457 boiled and denatured with denaturant, lane 5: gB1-705 treated with 1ng (1: 500) of trypsin at 37 ℃ for 1 hour and boiled and denatured with denaturants, lane 6: gB1-705, lane 7: gB1-705 treated with 100ng (1: 5) of trypsin at 37 ℃ for 3 hours and boiled and denatured with denaturants, lane 8: gB1-705 treated with 1ng (1: 500) of chymotrypsin at 37 ℃ for 1 hour and boiled and denatured with denaturant, lane M: BenchMark prestained Ladder and lane M': MagicWestern standard.
A band around about 300kDa was detected when gB1-705 was analyzed by SDS-PAGE under non-reducing and unboiled conditions (lane 1). Boiling of gB1-705, on the other hand, detected a band around 100kDa (lane 2). It means that the trimer and the monomer of gB are changed from trimer to monomer by boiling, respectively, and thus it is not shown that the trimer is formed through S-S bond.
gB is fragmented by treatment with trypsin or chymotrypsin, respectively. The reactivity of these gB with the antibody E7 scFv-hFc was examined by Western blotting. The E7 scFv-hFc showed reactivity with non-reduced and unboiled gB1-705 (lane 1), boiled gB1-705 (lane 2), reduced and boiled gB1-705 (lane 3) and protease treated gB fragments ( lanes 5, 6, 7) (FIG. 1). The same reaction pattern was observed for antibodies E17 and E31 (table 1). These results indicate that the antibodies E7, E17 and E31 scFv-hFc are contiguous epitopes. Antibodies a17, D1, D2, D3, D37, D48 and E15 scFv-hFc showed reactivity with non-reduced and unboiled gB1-705, boiled gB1-705, reduced and boiled gB1-705, and gB fragments treated with protease, but their reactivity with gB fragments showed patterns different from those of E7, E17, E31 (table 1). The same pattern of reaction between D1 and D2, and between D3 and D37, indicates that the same or nearby epitopes are recognized, respectively.
Groups can be divided into 6 according to the pattern of reactivity of the gB fragments. E41, F13, F18, F19, F22, F30, and F78scFv-hFc showed reactivity only with non-reduced and unboiled gB1-705 (table 1). These antibody clones are shown to be trimer-specific antibodies and to be discrete epitopes. F7, F11, F12, F33, F52, F65, F67, F68, F69, F76, F80, F87, G39, G64, G76, H15, H34, H57, H61, H65, G10, G25 and G65 show reactivity not only with non-reduced and unboiled gB1-705 but also with boiled gB1-705 (table 1). Indicating that these antibody clones are not trimer-specific antibodies but are discrete epitopes recognizing some degree of steric structure. E8, E35, E82 and E88 showed reactivity with non-reducing and non-boiling gB1-705, boiling gB1-705 and reducing and boiling gB1-705, but reactivity with protease fragments was not confirmed (Table 1). These antibody clones are reactive with gB in a fully denatured state and thus represent a continuous epitope, but regions of it may be susceptible to proteases. It was found that the 44 anti-gB antibody clones obtained by western blot analysis using gB and gB fragments as described above were divided into 9 groups. It was confirmed that 30 clones were discontinuous epitopes and 14 clones were continuous epitopes.
[ Table 1]
TABLE 1 reaction patterns of gB1-705 and fragments thereof in anti-gB antibodies
Figure BDA0002392880460000191
The opposite side is 1 +; -is reactive, -; non-reactive, 2ME +; with 2-mercaptoethanol treatment, boil +; with boiling treatment
The second phase 2 is treated with trypsin and chymotrypsin. The pattern of reactivity of fragmented gB with antibodies was divided into A, B, C, D, E, F6 patterns.
< Classification of antibodies by competitive ELISA >
Furthermore, to classify the obtained 44 antibody clones, competition ELISA using single-chain antibody phage against gB clone and scFv _ hFc was performed.
The competition between the resulting single-chain antibody phage and scFv-hFc was evaluated by ELISA. The gB1-705 was diluted to 1. mu.g/mL with PBS, 50. mu.L was added to the MaxiSorp plate, and the mixture was incubated overnight at 4 ℃ to immobilize gB 1-705. After immobilization, the plate was washed with PBS, 50. mu.L of scFv-hFc was added to the wells of the plate, and incubated at 25 ℃. After 1 hour, 50. mu.L of single-chain antibody phage was added to the wells of the plate and incubated at 25 ℃. After 1 hour, the plates were washed with PBST, and anti-M13/HRP 100. mu.L of the detection antibody was added to the wells of the plates and incubated at 25 ℃. After 1 hour, the plate was washed with PBST, and 100. mu.L of TMB was added to the wells of the plate to develop color. After 30 minutes, the reaction was terminated with 1N sulfuric acid, and the absorbance (O.D.450nm/650nm) was measured with a microplate reader.
Based on the results, a correlation chart (graph) of the antibody was prepared2)。ConfirmationThe group including antibodies E41, F18, F19, F13, F78, F22, and F30 was a group showing reactivity only to non-reduced and non-boiled gB1-705, but was classified into a group of antibodies E41, F18, F19, F13, and F78 and a group of antibodies F22, and F30 according to the results of competition ELISA, unlike the other groups. On the other hand, the other groups are not further subdivided from the above-mentioned groups. The anti-gB clones were therefore divided into 10 groups in total. In addition, D48 is reactive with gB370-457, thereby indicating the presence of epitopes in 370aa to 457aa of gB.
Example 3 epitope identification of gB antibodies using alanine scanning
Genes in which charged amino acid residues (at 187) in 1 to 705aa, which are extracellular domains of gB, were modified to alanine, respectively, were constructed by PCR and cloned into pCAGGS 1-dhfr-neo. For expression, FreeStyle293 or Expi293 expression systems were used. The expression level of the resulting gB alanine substitution and the binding activity of the alanine substitution to the antibody fragment were evaluated by ELISA.
The culture supernatant containing the gB alanine substitution was added to a MaxiSorp plate, and the mixture was incubated at room temperature for 1 hour to immobilize the gB alanine substitution. After immobilization, the plate was washed with PBST, and 100. mu.L of detection antibody streptococcin (Streptactin)/HRP (IBA) was added to the wells of the plate, followed by incubation at room temperature. After 1 hour, the plate was washed with PBST and developed by adding 100. mu.L of TMB to the plate wells. After 30 minutes, the reaction was terminated with 1N sulfuric acid, and the absorbance (O.D.450nm/650nm) was measured with a microplate reader to determine the expression level.
On the other hand, the culture supernatant containing the gB alanine substitution was added to the streptococcin-immobilized maxisorplate, and the mixture was incubated at room temperature for 1 hour to immobilize the gB alanine substitution. After immobilization, the plate was washed with PBST, and 100. mu.L of the antibody fragment was added to the well of the plate, followed by incubation at room temperature. After 1 hour, the plate was washed with PBST, 100. mu.L of detection antibody anti-human Fc/HRP (BIO COSMO) was added to the wells of the plate, and incubated at room temperature. After 1 hour, the plate was washed with PBST, and 100. mu.L of TMB was added to the wells of the plate to develop color. After 30 minutes, the reaction was terminated with 1N sulfuric acid, and the color development value (O.D.450nm/650nm) was measured with a microplate reader to determine the binding activity. Candidate epitopes were selected by the presence or absence of a change in reactivity per unit expression compared to wild-type gB1-705 without alanine substitution. Further, for antibodies that need to be accurately detected, the range of candidate epitopes was narrowed by ELISA using purified products of gB alanine substitutions.
The epitope of each antibody was predicted based on the binding activity of the alanine substitution to the antibody fragment, and the results are shown in table 2. The antibody directed against domain IV forming the top part of the gB trimer had 28 clones, the number being the highest. Next to this, there were 8 clones for the antibody directed against domain I forming the bottom part, while there were only 1 clone for the antibody directed against domain II forming the middle part. In the antibody for realizing epitope identification, epitopes are mostly located at positions dispersed from each other on the amino acid sequence. The positions of the gBs of HSV1 were confirmed in MOE (FIG. 3) using the gB of HSV2 as a template, as structurally close amino acids (data not shown). For D48, which uniquely recognizes domain II, the epitopes were identified as R391 and D362, but D48 reacted with gB37-457, so R391 is likely to be critical for the epitope. Furthermore, epitopes of 7 clones (E82, E88, F7, F65, F80, G10, and G76) out of 44 clones were not identified, and clones whose epitopes were identified were classified in the same group based on the grouping by the prior competition ELISA or the like, and therefore, the possibility that amino acid residues in the vicinity thereof were epitopes was high. For example, epitopes of E82 and E88, while not identified, are known as R532, R613, H632 for E8 and E35. E82 and E88 compete with E8 and E35 in a competition ELISA, and therefore the epitopes of E82 and E88 are presumed to be in the vicinity of R532, R613, and H632. It is considered that since this analysis is limited to charged amino acid residues, the epitope of the antibody whose epitope is not identified is likely to be an amino acid residue other than the charged amino acid in this meaning.
[ Table 2]
TABLE 2 epitope identification of anti-gB antibodies by alanine scanning
Figure BDA0002392880460000221
ND: not detected out
Example 4 analysis of gB using a neutralization assay for the presence of neutralizing and non-neutralizing epitopes in the top and bottom portions
< cells and viruses >
Vero cells (ccl.81) were purchased and used from ATCC in virus culture, infection titer assay, and neutralization antibody titer assay. Human herpes virus 2(HSV-2) MS strain [ VR-540] was used in neutralization assays and in anti-infective ability assays. For HSV-1, the KOS strain (VR-1493) was purchased and used from the ATCC. Culturing Vero cells in MEM medium containing 10% FBS to whole pieces, inoculating HSV-2MS strain and HSV-1KOS strain with m.o.i. 0.01-1, culturing in MEM medium containing 2% FBS for 2-3 days, and then releasing virus in cells into the culture medium by 3 times of freezing and thawing. After centrifugation, the supernatant was recovered as HSV-2 and HSV-1 virus pools.
< Virus neutralization assay >
The neutralizing activity of the resulting anti-gB clones was evaluated by a virus neutralization assay. The neutralization test was carried out by using 2 methods of plaque number reduction activity (plaque reduction activity) assay and intercellular infection spread inhibition activity assay. The target viruses used were 2 types, HSV-2MS strain and HSV-1KOS strain. For the plaque reduction activity assay, the test antibody was adjusted to a predetermined concentration, mixed with HSV-2MS strain or HSV-1KOS strain at about 100PFU, and reacted at 37 ℃ for 1 hour. The reaction solution was inoculated onto an entire piece of Vero cells in a 48-well plate, adsorbed at 30 ℃ for 1 hour, cultured in a medium containing 1% methylcellulose for 24 hours, and then inactivated and fixed at-20 ℃ for 30 minutes using 50% methanol/50% ethanol (-20 ℃) obtained by mixing methanol and ethanol in a ratio of 1: 1. Then, the anti-HSV gB monoclonal antibody was reacted at 37 ℃ for 1 hour, immunostaining was performed using anti-mouse IgG-HRP (Dako P0447) and TMBH (MOS TMBH-1000), an image of each well was captured using an ELISPOT analyzer, and the number of plaques was counted using analysis software.
For intercellular infection spread inhibitory activity assay, an entire piece of Vero cells was inoculated with HSV-2MS strain or HSV-1KOS strain of about 100PFU in a 48-well plate, adsorbed at 30 ℃ for 1 hour, then added with 1% methylcellulose medium containing the test antibody at a prescribed concentration (antibody concentrations of 5. mu.g/mL, 25. mu.g/mL, and 125. mu.g/mL), cultured for about 40 hours for the HSV-2MS strain, cultured for about 48 hours for the HSV-1KOS strain, and then inactivated and fixed with 50% methanol/50% ethanol (-20 ℃) at-20 ℃ for 30 minutes. Then, the anti-HSV gB monoclonal antibody at 37 ℃ reaction for 1 hours, with anti-mouse IgG-HRP (DakoP0447) and TMBH immunostaining, use ELISPOT analyzer capture each hole of the image, analysis of plaque size average value using analysis software.
The results are shown in Table 3. The obtained 44 clones are roughly classified into two types, namely, a neutralizing antibody and a non-neutralizing antibody, and the neutralizing antibody includes a clone that neutralizes both HSV-1 and HSV-2, a type-specific clone that neutralizes only either one of them, a clone that has a strong plaque-reducing activity but does not have an intercellular infection spread inhibitory activity (the intercellular infection spread inhibitory activity is weak), a clone that has both a strong plaque-reducing activity and an intercellular infection spread inhibitory activity, and the like. It was confirmed that the neutralization pattern is correlated with the grouping result (table 3, fig. 2).
[ Table 3]
Table 3: neutralizing Activity of anti-gB antibodies
Figure BDA0002392880460000251
N.T.: not tested
Plaque number-reducing activity was analyzed at 5. mu.g/mL, 25. mu.g/mL and 125. mu.g/mL, and those having neutralization ability at 5. mu.g/mL or less were designated "+", those having neutralization ability at 25. mu.g/mL or less were designated "+", and those having neutralization ability at 125. mu.g/mL or less were designated "+". The intercellular infection spread inhibition was analyzed at 5. mu.g/mL, 10. mu.g/mL and 20. mu.g/mL, and those with neutralization ability observed at 5. mu.g/mL or less were designated "+", those with neutralization ability observed at 10. mu.g/mL or less were designated "+", and those with neutralization ability observed at 20. mu.g/mL or less were designated "+". The measurements were performed in duplicate experiments (duplicate).
According to table 2, although the proportion of the antibodies against domain IV and domain I on gB2 antigen was large in the whole antibody, the antibodies against domain IV and domain I, although partially having neutralizing activity, were mostly non-neutralizing antibodies that did not exhibit neutralizing activity (table 3). On the other hand, the antibody against domain II is a neutralizing antibody although the proportion of the antibody in the whole antibody is small. The location of these epitopes is shown in the MOE diagram (figure 3), the epitope identification result for each gB antibody. These results show that neutralizing epitopes and non-neutralizing epitopes are present in domain IV and domain I, neutralizing epitopes are present in domain II, and more "prominent" epitopes are present in domain IV and domain I.
Example 5 design and evaluation of vaccine antigens
< strategy for design of modified gB antigen >
As a result of the extensive availability of anti-HSV 2-gB antibodies and their epitope mapping and functional classification, it was known that neutralizing and non-neutralizing epitopes were present in domain IV and domain I, and neutralizing epitopes were present in domain II on gB2 antigen. From the viewpoint of exhibiting defensive activity, a modified gB antigen was designed based on the use of a neutralizing epitope as a beneficial epitope, the use of a non-neutralizing epitope as a non-beneficial or harmful epitope, and the deipitopic formation of the non-neutralizing epitope present in gB domain I and domain IV. The basic wild-type gB is extracellular domain gB1-705, and when used for streptococcin purification, a streptokinase tag II is added to the C-terminal side (fig. 4 (a)).
First, gB modification was performed by adding N-type sugar chains. The N-type sugar chains are different from the O-type sugar chains, and are assigned to the sequence of NXT or NXS (X is an arbitrary amino acid other than proline) as a consensus sequence. In general, it is difficult to produce an antibody to a glycopeptide, and since the sugar chain is bulky, it is also difficult to produce an antibody to the periphery (non-patent document 16). It is believed that domain IV of HSV2-gB is a region important for binding to the receptor, but is located furthest from and exposed at the surface of the viral membrane, and thus the antibody readily binds thereto. Indeed, the above results have shown that there are more antibodies recognizing domain IV in human serum. Therefore, it was determined that N-type sugar chains were introduced into 3 non-neutralizing epitopes of the domain IV (FIG. 4B and FIG. 5). On the other hand, domain I is located at the root of gB and is a region important for fusion with host cells, but in the case where only the extracellular domain is expressed, a region originally in contact with the surface of the viral membrane is exposed on the surface. In addition, vaccine antigen design using only the extracellular domain of gB in the manufacture of vaccines is envisaged. Therefore, there is a possibility that the region in contact with the viral membrane is newly exposed, and 1 of these non-neutralizing epitope regions is introduced with an N-type sugar chain (FIG. 4B and FIG. 5). When HSV-1gB (PDB No.3NWF) is used as a reference, sugar chains originally added to wild-type gB are located at N115, N371, and N649 ((B) of FIG. 4). D199, R567, R602, and S631 were selected as new sugar chain introduction positions.
This is followed by the modification of gB by alanine exchange. The epitope of an antibody often contains charged amino acid residues. Therefore, a de-epitopic method may be used in which a charged amino acid residue is replaced with an uncharacterized amino acid residue. This method has an advantage that epitope removal can be accurately performed, unlike the introduction of an N-type sugar chain. In domain IV, a neutralizing epitope is present in addition to the non-neutralizing epitope, and it is determined that 1 alanine substitution (R613A) is performed (fig. 4(B) and fig. 5).
< preparation of modified gB1-705 >
cDNA (SEQ ID NO: 4) of the extracellular domain of gB (1-705aa) from 333 strain of wild-type HSV-2 was cloned into pCAGGS 1-dhfr-neo. Designed with Strep TagII added to the C-terminus of gB. A modified form having the following mutations introduced therein was designed using this sequence as a template.
Modified body bcev 1-3: D199N, D200A, H201T
Modification bceg 13: R567N, P568S, G569S
Modifier bcev 11: D199N, D200A, H201T, R613A
Modifier bcev 12: D199N, D200A, H201T, R567N, P568S, G569S, R613A
Modifier bcev 13: D199N, D200A, H201T, R567N, P568S, G569S, R613A, S631N, H632A, Q633T
Modifier bcev 19: D199N, D200A, H201T, R567N, P568S, G569S, R613A, S631N, H632T, Q633T
Modified body bcev 19': R567N, P568S, G569S, R613A, S631N, H632T, Q633T
Modifier bcev 50: D199N, D200A, H201T, R567N, P568S, G569S, R613A, R602N, D603A, A604T, S631N, H632T, Q633T
Modified body bcev 50': R567N, P568S, G569S, R613A, R602N, D603A, A604T, S631N, H632T, Q633T
For expression, FreeStyle293 or Expi293 expression systems were used. The expression plasmid was transfected into cells, and the culture supernatant was recovered at 4 to 6 days. In order to suppress the influence of biotin contained in the medium, the culture supernatant containing gB was concentrated with a UF membrane. The concentrated culture supernatant was purified using a StrepTactin column to obtain purified gB.
The properties of the purified gB1-705 thus obtained were confirmed to be multimeric by SDS-PAGE and gel filtration chromatography. For gel filtration chromatography, each modified gB purified product was loaded at a concentration of 100. mu.g/mL using Superdex200 Increate 5/150GL (GE healthcare) as a column. The flow rate was 0.4 mL/min, and D-PBS was used as the electrophoresis buffer to detect A280.
The modified sites of the 6 types of modified gB and their characteristics are shown in Table 4.
[ Table 4]
Figure BDA0002392880460000281
N.T.: not tested
The modified bcev1-3 containing D199N, D200A and H201T was a type to which a mutation for introducing a sugar chain into the domain I was added, and the expression level was increased by about 2 times as compared with the wild-type gB 1-705. It is considered that this region is originally stabilized in a state of being in contact with the surface of the virus membrane. It is presumed that the wild-type gB1-705 is unstable because the region which is not originally exposed to the solvent is exposed by secretory expression, and bcev1-3 is stabilized by adding a sugar chain thereto, resulting in an increase in the expression level.
bceg13 is a modified gB with the addition of R567N, P568S, G569S to domain IV. The expression level of bcev1-3 was changed to 0.3-fold in comparison with wild-type gB 1-705. Since the trimer structure is maintained as in the wild type, the structure is maintained, and not a dangerous mutation, but the single-strand allosteric folding speed may be slower than that of the wild type.
bcev11 is a modified form of bcev1-3 in which mutations of D199N, D200A and H201T are added and R613A located in domain IV is introduced. The expression level of bcev11 was comparable to that of wild-type gB 1-705.
Considering that the expression level is increased by the mutation of bcev1-3, the mutation of R613A may be a mutation which reduces the expression level or is equivalent to the wild type.
The trait of bcev11 is the same as wild type, forming trimers.
bcev12 is a modified gB in which R567N, P568S, G569S were further added to bcev 11. According to the results of bceg13, the addition of mutations R567N, P568S, G569S should lead to a decrease in expression, but unexpectedly to an increase of 2-fold compared to the wild type. This expression level was comparable to that of bcev1-3 modified with D199N, D200A and H201T alone. The mutation of R613A also helps to reduce the expression level, but probably because R567N, P568S and G569S are also added, so that the structure and folding speed of the domain IV are close to bcev 1-3.
bcev19 is a gB modifier in which mutations of S631N, H632T, and Q633T have been introduced into bcev 12. The expression level of bcev19 was 0.6-fold compared with the wild type, and it contained not only trimer but also a trace amount of multimer. It was also investigated to introduce another mutation into S631, H632, Q633 or to introduce a sugar chain into a nearby position other than 631-633aa, but no significant improvement effect was seen in terms of properties (data not shown).
bcev50 is a gB modifier in which mutations of R602N, D603A, and a604T are introduced into domain IV of bcev 19. The expression level was further reduced compared with that of bcev19, and was 0.3-fold higher than that of the wild type. In the case of traits, it also contains a trimer and a multimer as in the case of bcev 19.
< test for binding Activity of modified gB1-705 >
In order to confirm that the reactivity with the targeted non-neutralizing antibody was reduced or eliminated, the reactivity of the prepared modified bodies bcev1-3, bceg13, bcev12, bcev19 and bcev50 to the anti-gB 2 monoclonal antibody of 44 clones was evaluated by ELISA. As a control, wild-type gB1-705 was used. The gB1-705 was diluted to 1. mu.g/mL with PBS, 50. mu.L was added to MaxiSorplate, and the mixture was incubated overnight at 4 ℃ to immobilize gB 1-705. After immobilization, the plate was washed with PBS, and 100. mu.L of the obtained antibody was added to the well of the plate, followed by incubation at 37 ℃. After 1 hour, the plate was washed with PBST, 100. mu.L of detection antibody anti-human IgG Fc/HRP (ROCKLAND) was added to the wells of the plate, and incubated at 37 ℃. After 1 hour, the plate was washed with PBST, and 100. mu.L of TMB was added to the wells of the plate to develop color. After 30 minutes, the reaction was terminated with 1N sulfuric acid, and the absorbance (O.D.450nm/650nm) was measured with a microplate reader.
The results are shown in Table 5.
[ Table 5]
TABLE 5 reactivity of modified gB with the antibodies obtained
Figure BDA0002392880460000311
N.T.: not tested
bcev1-3 had no reactivity with F13, F19, and F78 having D200 and H201 as epitopes by introducing mutations of D199N, D200A, and H201T, and bceg13 had no reactivity with F76 and G25 having R567 as epitopes by introducing mutations of R567N, P568S, and G569S.
In addition, in bcev12 into which modification of bcev1-3, modification of bceg13 and R613A were introduced, all of the reactivity of the antibody with each mutation position as an epitope was lost.
Bcev19, which introduced a mutation of S631N, H632A or Q633T into bcev12, maintained the reaction with 22 kinds of neutralizing antibodies while the reaction with non-neutralizing antibodies was reduced.
Of the 21 non-neutralizing antibodies, 8 species of recognition domains IV F7, F65, F68, F80, and G76 and recognition domains I F18, F19, and F78 remained reactive.
The reactivity of bcev50, in which mutations of R602N, D603A and a604T were introduced into bcev19, with all 21 non-neutralizing antibodies was lost. On the other hand, reactivity with 14 out of 23 neutralizing antibodies was maintained, but reactivity with 9 was reduced.
These results probably imply that immunization bcev50 resulted in less induction of non-neutralizing antibodies and that induction of neutralizing antibodies was slightly more difficult. The difference between bcev19 and bcev50 is whether there is a mutation in domain IV of R602N, D603A, a 604T. Nevertheless, differences in reactivity with antibodies directed against domain I are also generated. By modifying domain IV, the trimer is maintained but the structure of domain I may have changed. In conclusion, bcev19 and bcev50 are not able to form non-neutralizing antibodies as compared with gB1-705, which is a wild-type antigen, and are expected to be novel vaccine antigens that can induce a desirable immune response.
< mouse immunogenicity test of bcev19 and bcev50 >
The prepared modified gB antigens bcev19 and bcev50 were subjected to mouse immunogenicity tests, respectively. In each experiment, the antigen was administered in amounts of 0.3. mu.g/mouse and 1. mu.g/mouse 3 times subcutaneously at 2-week intervals. The experiment was performed with the number of animals in each group being 4.
Immunogenicity tests of modified gB antigens were performed using wild-type gB1-705(gB WT) as positive controls and saline as negative controls. A prescribed amount of antigen was dissolved in physiological saline (saline) for injection, and BALB/c mice (5 weeks old, female) were immunized 3 times in a volume of 200. mu.L/mouse in total at 2-week intervals together with MPLA (10. mu.g/mouse) and CpG (1. mu.g/mouse) for each mouse, subcutaneously. Blood was collected from each individual 2 weeks after the last immunization (3 rd), and serum was prepared. The prepared sera were diluted in a gradient and evaluated for binding antibody titer to wild-type gB antigen (anti-gB ELISA) and neutralizing antibody titer to HSV-2 (activity of 50% reduction in plaque number).
The results of bcev19 are shown in FIG. 6, and the results of bcev50 are shown in FIG. 7. Mean values for n-4 are plotted and ± SE error bars are labeled. As a result of evaluating the binding antibody-inducing activity (anti-gB ELISA) against the wild-type gB antigen and the neutralizing antibody-inducing activity (plaque number reduction rate) against HSV-2 using sera collected 2 weeks after the last immunization, it was confirmed that bcev19, bcev50 induced higher neutralizing antibody activity at either administration amount with the binding antibody activity less than that of the wild-type gB antigen (gB 1-705).
It is considered that this result indicates that an immune response against the remaining neutralizing epitope (beneficial epitope) can be efficiently and effectively induced by deipitopicating the non-neutralizing epitope (harmful/useless epitope) on the wild-type gB antigen by adding an N-type sugar chain and substituting alanine. In other words, it can be said that the deviated immune response (immune deviation) against the wild-type gB antigen can be corrected (immune correction) to an ideal form by the immune refocusing (immunological refocusing) strategy of the present inventors.
< mice infection test of bcev19 and bcev50 >
The mouse genital herpes infection model was used to evaluate the anti-infective ability of the modified gB antigens bcev19 and bcev50, respectively, under prophylactic administration. In either experiment, wild-type gB1-705(gBWT) was used as a positive control. The antigen was immunized subcutaneously 3 times at intervals of 0.03. mu.g/mouse, 0.1. mu.g/mouse, 0.3. mu.g/mouse, 1. mu.g/mouse and 2 weeks. To increase the infection efficiency at the time of virus inoculation performed 2 weeks after the last immunization (3 rd), Depo-Provera was subcutaneously inoculated at 2 mg/virus 6 days before virus inoculation. Under anesthesia at 5X 105PFU/20. mu.L/vaginal inoculation of HSV-2MS strain alone, 21 days follow-up observation. The anti-infective ability was evaluated by using the survival days (survival rate) and symptom score as indicators. Regarding the symptom score, scores of 3 stages and 2 stages were classified and set according to the degree of vaginal lesion symptoms and general symptoms, respectively. The symptom score was obtained by adding the scores of vaginal lesions and systemic symptoms shown below. Scores for vaginal lesions (0: no change, 1: localized erythema and swelling, 2: extensive swelling and edema, 3: ulceration and bleeding). Scores for systemic symptoms (0: no change, 1: piloerection, 2: hind limb paralysis). In addition, the score of the dead or sacrificed individual was set to 6. The experiment was performed with the number of animals in each group being n-10, and the average value of these was plotted in the figure.
The results of bcev19 are shown in table 6 (days to live), figure 8 (survival rate) and figure 9 (symptom score). Both gB WT and bcev19 showed significant effect of extending the number of days to live compared to the negative control group (saline group) at all the doses set (0.03 to 1 μ g/dose), the median number of days to live (MST) of the gB WT group showed no clear dose dependence, and the MST ratio to the saline group remained less than 2 at any dose, whereas MST of the bcev19 group almost all showed dose dependence, and the MST ratio was >2.8 at 3 doses of 0.1 μ g/dose or more, and clear improvement in survival rate was seen (table 6, fig. 8). With respect to the symptom score, the gB WT administration group did not show clear dose dependence, with severe symptoms at the highest dose, i.e., 1 μ g/dose, whereas the bcev19 administration group showed dose-dependent and significant improvement effect (fig. 9).
[ Table 6]
Figure BDA0002392880460000341
MsT: mean days of survival ×: p < 0.0001/. + -: 0.0001 < p < 0.001/: p is more than 0.001 and less than 0.01(Kaplan-Meier method)
The results of bcev50 are shown in table 7 (days to live), fig. 10 (survival rate) and fig. 11 (symptom score). Like bcev19, bcev50 also showed clear superiority in survival days, survival rates, and any index of symptom scores as compared with gB WT.
[ Table 7]
Figure BDA0002392880460000342
MST: mean days of survival ×: p < 0.0001/. + -: 0.0001 < p < 0.001/: p is more than 0.001 and less than 0.01(Kaplan-Meier method)
< analysis of immune refocusing >
For the modified gB antigens bcev19 and bcev50, which were found to be superior to the wild-type gB (gB1-705) in the mouse immunogenicity test and the mouse anti-infection test, whether or not immune refocusing was induced was analyzed by ELISA immobilized with gB1-457 and gB111-457, respectively.
Fig. 12 shows the analysis result of bcev19, and fig. 13 shows the analysis result of bcev 50. It was confirmed that both of the bcev19 immune serum and the bcev50 immune serum had higher binding antibody activity against gB1-457 and gB111-457 than the wild-type gB immune serum. The results are believed to indicate that: by removing the non-neutralizing epitope mainly present in domain IV, which is considered to be a decoy region in the wild-type gB antigen, by adding an N-type sugar chain and substituting alanine, it is possible to induce an immune response against the neutralizing epitope (beneficial epitope) mostly remaining in domains I and II more efficiently and effectively. In other words, it can be said that the biased immune response (immune bias) against the decoy region on the wild-type gB antigen can be corrected (immune corrected) to the ideal form by the immune refocusing strategy.
< Effect of N-type sugar chain introduced into gB Domain I >
Bcev19, which is a gB modified form, has D199N, D200A, and H201T introduced into domain I, and modifications of R613A, R567N, P568S, G569S, S631N, H632T, and Q633T introduced into domain IV. Further, D199N, D200A and H201T were introduced into domain I of bcev50, and modifications of R613A, R567N, P568S, G569S, S631N, H632T, Q633T, R602N, D603A and a604T were introduced into domain IV. To further examine the effect of the modifications introduced into bcev19 and bcev50, we created modifications, i.e., bcev19 'and bcev 50', which restored only the modifications D199N, D200A and H201T of domain I contained in bcev19 and bcev50, respectively, to the original amino acid sequences.
Overexpression was performed using the Expi293 expression system, and the expression levels were compared (Table 8). As a result, the expression levels of bcev19, bcev19 ', bcev50 and bcev 50' were 10.81. mu.g/mL, 1.28. mu.g/mL, 6.29. mu.g/mL and 4.36. mu.g/mL, respectively. Comparison of the expression levels of bcev19 and bcev19 'indicates that bcev19 is 8.45 times higher than bcev 19', and comparison of the expression levels of bcev50 and bcev50 'indicates that bcev50 is 1.44 times higher than bcev 50'. This means that D199N, D200A and H201T in bcev19 and bcev50 are mutations contributing to increase in expression level. The same results were obtained for bceg1-3 in which each of gB1-705 was modified with D199N, D200A and H201T, and this was supported (Table 4).
[ Table 8]
Figure BDA0002392880460000361
Further, the gel filtration chromatography was used to analyze the properties of bcev19, bcev19 ', bcev50 and bcev 50'. The results are shown in fig. 14. bcev19 is higher than the native trimer of bcev19 '(FIG. 14(A)), and further bcev50 is higher than the trimer of bcev 50' (FIG. 14 (B)). This means that D199N, D200A, and H201T in bcev19 and bcev50 are mutations contributing to the improvement of the trait.
Then, comparison was made between bcev19 and bcev19 ', bcev50 and bcev 50', respectively, in a mouse immunogenicity test. 3 subcutaneous immunizations were performed at 2-week intervals with 0.3. mu.g/mouse and 1. mu.g/mouse of antigen. The number of animals in each group was set to n-4. A comparison of bcev19 and bcev19 'is shown in fig. 15, and a comparison of bcev50 and bcev 50' is shown in fig. 16 (the average value of n ═ 4 is plotted in the figure and ± SE error bars are shown). Serum collected 2 weeks after the last immunization was used to evaluate the binding antibody-inducing activity against the wild-type gB antigen (anti-gB ELISA) and the neutralizing antibody-inducing activity against HSV-2 (plaque number reduction rate), and as a result, it was found that, in the case of either index, bcev19 tended to exhibit higher antibody-inducing ability than bcev19 'and bcev50 tended to exhibit higher antibody-inducing ability than bcev 50'.
From the above results, it was found that the N-type sugar chains of domain I (D199N, D200A, and H201T) introduced into the mutations of the modified bodies bcev19 and bcev50 are mutations contributing not only to enhancement of the neutralizing antibody-inducing ability but also to enhancement of the protein expression level and improvement of the properties.
Industrial applicability
The modified HSV gB protein can be used for preparing vaccines which are effective in preventing and treating HSV infection.
Sequence listing
<110> KM Biomedicine pinch company (KM Biologics Co., Ltd.)
<120> modified HSV gB protein and HSV vaccine comprising same
<130>FP18-0825-00
<150>JP2017-165684
<151>2017-08-30
<160>4
<170>PatentIn version 3.5
<210>1
<211>705
<212>PRT
<213> Herpes simplex virus-2 (Herpes simplex virus-2)
<220>
<223>HSV gB1-705
<400>1
Ala Pro Ala Ala Pro Ala Ala Pro Arg Ala Ser Gly Gly Val Ala Ala
1 5 10 15
Thr Val Ala Ala Asn Gly Gly Pro Ala Ser Arg Pro Pro Pro Val Pro
20 25 30
Ser Pro Ala Thr Thr Lys Ala Arg Lys Arg Lys Thr Lys Lys Pro Pro
35 40 45
Lys Arg Pro Glu Ala Thr Pro Pro Pro Asp Ala Asn Ala Thr Val Ala
50 55 60
Ala Gly His Ala Thr Leu Arg Ala His Leu Arg Glu Ile Lys Val Glu
65 70 75 80
Asn Ala Asp Ala Gln Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr
85 90 95
Val Val Gln Phe Glu Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly
100 105 110
Gln Asn Tyr Thr Glu Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala
115 120 125
Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser
130 135 140
Gln Val Trp Phe Gly His Arg TyrSer Gln Phe Met Gly Ile Phe Glu
145 150 155 160
Asp Arg Ala Pro Val Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala
165 170 175
Lys Gly Val Cys Arg Ser Thr Ala Lys Tyr Val Arg Asn Asn Met Glu
180 185 190
Thr Thr Ala Phe His Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys
195 200 205
Pro Ala Lys Val Ala Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp
210 215 220
Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala Phe His Arg Tyr Gly Thr
225 230 235 240
Thr Val Asn Cys Ile Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro
245 250 255
Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro
260 265 270
Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala
275 280 285
Ala Asp Arg Phe Lys Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr
290 295 300
Thr Lys Ala Arg Ala Thr Ser Pro Thr ThrArg Asn Leu Leu Thr Thr
305 310 315 320
Pro Lys Phe Thr Val Ala Trp Asp Trp Val Pro Lys Arg Pro Ala Val
325 330 335
Cys Thr Met Thr Lys Trp Gln Glu Val Asp Glu Met Leu Arg Ala Glu
340 345 350
Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp Ala Ile Ser Thr Thr Phe
355 360 365
Thr Thr Asn Leu Thr Gln Tyr Ser Leu Ser Arg Val Asp Leu Gly Asp
370 375 380
Cys Ile Gly Arg Asp Ala Arg Glu Ala Ile Asp Arg Met Phe Ala Arg
385 390 395 400
Lys Tyr Asn Ala Thr His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu
405 410 415
Ala Thr Gly Gly Phe Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr
420 425 430
Leu Ala Glu Leu Tyr Val Arg Glu Tyr Met Arg Glu Gln Asp Arg Lys
435 440 445
Pro Arg Asn Ala Thr Pro Ala Pro Leu Arg Glu Ala Pro Ser Ala Asn
450 455 460
Ala Ser Val Glu Arg Ile Lys Thr Thr Ser Ser IleGlu Phe Ala Arg
465 470 475 480
Leu Gln Phe Thr Tyr Asn His Ile Gln Arg His Val Asn Asp Met Leu
485 490 495
Gly Arg Ile Ala Val Ala Trp Cys Glu Leu Gln Asn His Glu Leu Thr
500 505 510
Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro Asn Ala Ile Ala Ser Ala
515 520 525
Thr Val Gly Arg Arg Val Ser Ala Arg Met Leu Gly Asp Val Met Ala
530 535 540
Val Ser Thr Cys Val Pro Val Ala Pro Asp Asn Val Ile Val Gln Asn
545 550 555 560
Ser Met Arg Val Ser Ser Arg Pro Gly Thr Cys Tyr Ser Arg Pro Leu
565 570 575
Val Ser Phe Arg Tyr Glu Asp Gln Gly Pro Leu Ile Glu Gly Gln Leu
580 585 590
Gly Glu Asn Asn Glu Leu Arg Leu Thr Arg Asp Ala Leu Glu Pro Cys
595 600 605
Thr Val Gly His Arg Arg Tyr Phe Ile Phe Gly Gly Gly Tyr Val Tyr
610 615 620
Phe Glu Glu Tyr Ala Tyr Ser His Gln Leu Ser Arg Ala AspVal Thr
625 630 635 640
Thr Val Ser Thr Phe Ile Asp Leu Asn Ile Thr Met Leu Glu Asp His
645 650 655
Glu Phe Val Pro Leu Glu Val Tyr Thr Arg His Glu Ile Lys Asp Ser
660 665 670
Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg Arg Asn Gln Leu His Asp
675 680 685
Leu Arg Phe Ala Asp Ile Asp Thr Val Ile Arg Ala Asp Ala Asn Ala
690 695 700
Ala
705
<210>2
<211>904
<212>PRT
<213> Herpes simplex virus-1 (Herpes simplex virus-1)
<400>2
Met His Gln Gly Ala Pro Ser Trp Gly Arg Arg Trp Phe Val Val Trp
1 5 10 15
Ala Leu Leu Gly Leu Thr Leu Gly Val Leu Val Ala Ser Ala Ala Pro
20 25 30
Thr Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala Asn
35 40 45
Gly Gly Pro Ala Thr Pro Ala Pro Pro Pro Leu Gly Ala AlaPro Thr
50 55 60
Gly Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro
65 70 75 80
Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr
85 90 95
Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala Asn
100 105 110
Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu
115 120 125
Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu
130 135 140
Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys
145 150 155 160
Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly
165 170 175
His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val
180 185 190
Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg
195 200 205
Ser Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His
210 215 220
Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala
225 230 235 240
Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro
245 250 255
Ser Arg Val Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile
260 265 270
Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val
275 280 285
Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg
290 295 300
Glu Gly Ser His Thr Glu His Thr Thr Tyr Ala Ala Asp Arg Phe Lys
305 310 315 320
Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala
325 330 335
Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val
340 345 350
Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys
355 360 365
Trp Gln Glu Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe
370 375 380
Arg Phe Ser Ser Asp Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr
385 390 395 400
Glu Tyr Pro Leu Ser Arg Val Asp Leu Gly Asp Cys Ile Gly Lys Asp
405 410 415
Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Asn Ala Thr
420 425 430
His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Gln Ala Asn Gly Gly Phe
435 440 445
Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr
450 455 460
Val Arg Glu His Leu Arg Glu Gln Ser Arg Lys Pro Pro Asn Pro Thr
465 470 475 480
Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys
485 490 495
Thr Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His
500 505 510
Ile Gln Arg His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp
515 520 525
Cys Glu Leu Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys
530 535 540
Leu Asn Pro Asn Ala Ile Ala Ser Val Thr Val Gly Arg Arg Val Ser
545 550 555 560
Ala Arg Met Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val
565 570 575
Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser Ser Arg
580 585 590
Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp
595 600 605
Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg
610 615 620
Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg Tyr
625 630 635 640
Phe Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser
645 650 655
His Gln Leu Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp
660 665 670
Leu Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val
675 680 685
Tyr Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu
690 695 700
Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp
705 710 715 720
Thr Val Ile His Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Gly
725 730 735
Ala Phe Phe Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val
740 745 750
Val Met Gly Ile Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser
755 760 765
Ser Phe Met Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val
770 775 780
Leu Ala Gly Leu Ala Ala Ala Phe Phe Ala Phe Arg Tyr Val Met Arg
785 790 795 800
Leu Gln Ser Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu
805 810 815
Leu Lys Asn Pro Thr Asn Pro Asp Ala Ser Gly Glu Gly Glu Glu Gly
820 825 830
Gly Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg
835 840 845
Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Lys
850 855 860
Lys Lys Gly Thr Ser Ala Leu Leu Ser Ala Lys Val Thr Asp Met Val
865 870 875 880
Met Arg Lys Arg Arg Asn Thr Asn Tyr Thr Gln Val Pro Asn Lys Asp
885 890 895
Gly Asp Ala Asp Glu Asp Asp Leu
900
<210>3
<211>946
<212>PRT
<213> Herpes simplex virus-2 (Herpes simplex virus-2)
<400>3
Met Arg Gly Gly Gly Leu Ile Cys Ala Leu Val Val Gly Ala Leu Val
1 5 10 15
Ala Ala Val Ala Ser Ala Ala Pro Ala Ala Pro Ala Ala Pro Arg Ala
20 25 30
Ser Gly Gly Val Ala Ala Thr Val Ala Ala Asn Gly Gly Pro Ala Ser
35 40 45
Arg Pro Pro Pro Val Pro Ser Pro Ala Thr Thr Lys Ala Arg Lys Arg
50 55 60
Lys Thr Lys Lys Pro Pro Lys Arg Pro Glu Ala Thr Pro Pro Pro Asp
65 70 75 80
Ala Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu ArgAla His Leu
85 90 95
Arg Glu Ile Lys Val Glu Asn Ala Asp Ala Gln Phe Tyr Val Cys Pro
100 105 110
Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys
115 120 125
Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala Val Val
130 135 140
Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr
145 150 155 160
Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly His Arg Tyr Ser Gln
165 170 175
Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val Pro Phe Glu Glu Val
180 185 190
Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser Thr Ala Lys Tyr
195 200 205
Val Arg Asn Asn Met Glu Thr Thr Ala Phe His Arg Asp Asp His Glu
210 215 220
Thr Asp Met Glu Leu Lys Pro Ala Lys Val Ala Thr Arg Thr Ser Arg
225 230 235 240
Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val GluAla
245 250 255
Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp
260 265 270
Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp
275 280 285
Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr
290 295 300
Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys Gln Val Asp Gly Phe
305 310 315 320
Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala Thr Ser Pro Thr Thr
325 330 335
Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp Trp Val
340 345 350
Pro Lys Arg Pro Ala Val Cys Thr Met Thr Lys Trp Gln Glu Val Asp
355 360 365
Glu Met Leu Arg Ala Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp
370 375 380
Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Gln Tyr Ser Leu Ser
385 390 395 400
Arg Val Asp Leu Gly Asp Cys Ile Gly Arg Asp Ala Arg Glu Ala Ile
405 410 415
Asp Arg Met Phe Ala Arg Lys Tyr Asn Ala Thr His Ile Lys Val Gly
420 425 430
Gln Pro Gln Tyr Tyr Leu Ala Thr Gly Gly Phe Leu Ile Ala Tyr Gln
435 440 445
Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr Val Arg Glu Tyr Met
450 455 460
Arg Glu Gln Asp Arg Lys Pro Arg Asn Ala Thr Pro Ala Pro Leu Arg
465 470 475 480
Glu Ala Pro Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr Thr Ser
485 490 495
Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Arg
500 505 510
His Val Asn Asp Met Leu Gly Arg Ile Ala Val Ala Trp Cys Glu Leu
515 520 525
Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro
530 535 540
Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met
545 550 555 560
Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val Ala Pro Asp
565 570 575
Asn Val Ile Val Gln Asn Ser Met Arg Val Ser Ser Arg Pro Gly Thr
580 585 590
Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln Gly Pro
595 600 605
Leu Ile Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu Thr Arg
610 615 620
Asp Ala Leu Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe Ile Phe
625 630 635 640
Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser His Gln Leu
645 650 655
Ser Arg Ala Asp Val Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile
660 665 670
Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg
675 680 685
His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg
690 695 700
Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp Thr Val Ile
705 710 715 720
Arg Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Cys Ala Phe Phe
725 730 735
Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val Val Met Gly
740 745 750
Val Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser Ser Phe Met
755 760 765
Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val Leu Ala Gly
770 775 780
Leu Val Ala Ala Phe Phe Ala Phe Arg Tyr Val Leu Gln Leu Gln Arg
785 790 795 800
Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu Leu Lys Thr
805 810 815
Ser Asp Pro Gly Gly Val Gly Gly Glu Gly Glu Glu Gly Ala Glu Gly
820 825 830
Gly Gly Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg
835 840 845
Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Arg
850 855 860
Lys Lys Gly Thr Ser Ala Leu Leu Ser Ser Lys Val Thr Asn Met Val
865 870 875 880
Leu Arg Lys Arg Asn Lys Ala Arg Tyr Ser Pro Leu His Asn Glu Asp
885 890 895
Glu Ala Gly Asp Glu Asp Glu Leu Ala Arg Lys Lys Gly Thr Ser Ala
900 905 910
Leu Leu Ser Ser Lys Val Thr Asn Met Val Leu Arg Lys Arg Asn Lys
915 920 925
Ala Arg Tyr Ser Pro Leu His Asn Glu Asp Glu Ala Gly Asp Glu Asp
930 935 940
Glu Leu
945
<210>4
<211>2712
<212>DNA
<213> Herpes simplex virus-2 (Herpes simplex virus-2)
<400>4
atgcgcgggg ggggcttgat ttgcgcgctg gtcgtggggg cgctggtggc cgcggtggcg 60
tcggcggccc cggcggcccc ggcggccccc cgcgcctcgg gcggcgtggc cgcgaccgtc 120
gcggcgaacg ggggtcccgc ctcccggccg ccccccgtcc cgagccccgc gaccaccaag 180
gcccggaagc ggaaaaccaa aaagccgccc aagcggcccg aggcgacccc gccccccgac 240
gccaacgcga ccgtcgccgc cggccacgcc acgctgcgcg cgcacctgcg ggaaatcaag 300
gtcgagaacg ccgatgccca gttttacgtg tgcccgcccc cgacgggcgc cacggtggtg 360
cagtttgagc agccgcgccg ctgcccgacg cgcccggagg ggcagaacta cacggagggc 420
atcgcggtgg tcttcaagga gaacatcgcc ccgtacaaat tcaaggccac catgtactac 480
aaagacgtgaccgtgtcgca ggtgtggttc ggccaccgct actcccagtt tatggggata 540
ttcgaggacc gcgcccccgt tcccttcgag gaggtgatcg acaagattaa cgccaagggg 600
gtctgccgct ccacggccaa gtacgtgcgg aacaacatgg agaccaccgc gtttcaccgg 660
gacgaccacg agaccgacat ggagctcaag ccggcgaagg tcgccacgcg cacgagccgg 720
gggtggcaca ccaccgacct caagtacaac ccctcgcggg tggaggcgtt ccatcggtac 780
ggcacgacgg tcaactgcat cgtcgaggag gtggacgcgc ggtcggtgta cccgtacgat 840
gagtttgtgt tggcgacggg cgactttgtg tacatgtccc cgttttacgg ctaccgggag 900
gggtcgcaca ccgagcacac cagctacgcc gccgaccgct tcaagcaggt cgacggcttc 960
tacgcgcgcg acctcaccac gaaggcccgg gccacgtcgc cgacgacccg caacttgctg 1020
acgaccccca agtttaccgt ggcctgggac tgggtgccga agcgaccggc ggtctgcacc 1080
atgaccaagt ggcaggaggt ggacgagatg ctccgcgccg agtacggcgg ctccttccgc 1140
ttctcctccg acgccatctc gaccaccttc accaccaacc tgacccagta ctcgctctcg 1200
cgcgtcgacc tgggcgactg cattggccgg gatgcccgcg aggccatcga ccgcatgttt 1260
gcgcgcaagt acaacgccac gcacatcaag gtgggccagc cgcagtacta cctggccacg 1320
gggggcttcc tcatcgcgta ccagcccctc ctcagcaaca cgctcgccga gctgtacgtg 1380
cgggagtaca tgcgggagca ggaccgcaag ccccggaatg ccacgcccgc gccactgcgg 1440
gaggcgccca gcgccaacgc gtccgtggag cgcatcaaga ccacctcctc gatcgagttc 1500
gcccggctgc agtttacgta taaccacata cagcgccacg tgaacgacat gctggggcgc 1560
atcgccgtcg cgtggtgcga gctgcagaaccacgagctga ctctctggaa cgaggcccgc 1620
aagctcaacc ccaacgccat cgcctccgcc accgtcggcc ggcgggtgag cgcgcgcatg 1680
ctcggagacg tcatggccgt ctccacgtgc gtgcccgtcg ccccggacaa cgtgatcgtg 1740
cagaactcga tgcgcgtcag ctcgcggccg gggacgtgct acagccgccc cctggtcagc 1800
tttcggtacg aagaccaggg cccgctgatc gaggggcagc tgggcgagaa caacgagctg 1860
cgcctcaccc gcgacgcgct cgagccgtgc accgtgggcc accggcgcta cttcatcttc 1920
ggcgggggct acgtgtactt cgaggagtac gcgtactctc accagctgag tcgcgccgac 1980
gtcaccaccg tcagcacctt catcgacctg aacatcacca tgctggagga ccacgagttt 2040
gtgcccctgg aggtctacac gcgccacgag atcaaggaca gcggcctgct ggactacacg 2100
gaggtccagc gccgcaacca gctgcacgac ctgcgctttg ccgacatcga cacggtcatc 2160
cgcgccgacg ccaacgccgc catgttcgcg gggctgtgcg cgttcttcga ggggatgggg 2220
gacttggggc gcgcggtcgg caaggtagtc atgggagtag tggggggcgt ggtgtcggcc 2280
gtctcgggcg tgtcctcctt tatgtccaac cccttcgggg cgcttgccgt ggggctgctg 2340
gtcctggccg gcctggtcgc ggccttcttc gccttccgct acgtcctgca actgcaacgc 2400
aatcccatga aggccctgta tccgctcacc accaaggaac tcaagacttc cgaccccggg 2460
ggcgtgggcg gggaggggga ggaaggcgcg gaggggggcg ggtttgacga ggccaagttg 2520
gccgaggccc gagaaatgat ccgatatatg gctttggtgt cggccatgga gcgcacggaa 2580
cacaaggcca gaaagaaggg cacgagcgcc ctgctcagct ccaaggtcac caacatggtt 2640
ctgcgcaagc gcaacaaagc caggtactct ccgctccaca acgaggacga ggccggagac 2700
gaagacgagc tc 2712

Claims (23)

1. A modified HSV gB protein which is a modified protein of the envelope glycoprotein b (gB) of Herpes Simplex Virus (HSV) (modified HSV gB protein) which is a modified HSV gB protein modified as follows: at least 1 of the non-neutralizing antibody-inducing epitopes (non-neutralizing epitopes) present in domain IV and domain I of wild-type HSV gB is rendered non-functional as an epitope.
2. The modified HSV gB protein according to claim 1, wherein said non-neutralizing epitope is an epitope comprising at least 1 amino acid residue present in a region which is located at a distance of 1.5nm or less from an amino acid residue corresponding to arginine residue 567 (R567), arginine residue 602 (R602), serine residue 631 (S631), or aspartic acid residue 199 (D199) in the amino acid sequence described in SEQ ID NO. 1, on the surface of the crystal structure of the extracellular domain of wild-type HSV gB.
3. The modified HSV gB protein of claim 1 or 2, wherein the non-neutralizing epitope is an epitope comprising amino acid residues corresponding to R567, R602, S631 or D199 of the amino acid sequence of seq id No. 1.
4. The modified HSV gB protein of any one of claims 1 to 3, wherein said modification comprises a modification by substitution of an amino acid residue and/or deletion of an amino acid residue.
5. The modified HSV gB protein of claim 4, wherein said modification comprises modification by introducing a sugar chain by substitution or deletion of an amino acid residue.
6. The modified HSV gB protein according to any one of claims 1 to 5, wherein said modification comprises a modification for introducing a sugar chain at a position of at least 1 amino acid residue selected from the group consisting of amino acid residues corresponding to D199, R567, R602, and S631 in the amino acid sequence shown in SEQ ID NO. 1.
7. The modified HSV gB protein according to any one of claims 1 to 6, wherein said modification comprises a modification for introducing a sugar chain at a position of at least 2 amino acid residues selected from the group consisting of the amino acid residues corresponding to D199, R567, R602, and S631 in the amino acid sequence shown in SEQ ID NO. 1.
8. The modified HSV gB protein of claim 7, wherein said modification comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residues R567 and S631 in the amino acid sequence of SEQ ID NO. 1.
9. The modified HSV gB protein according to claim 8, wherein said sugar chain is introduced by substituting the amino acid residues R567N, P568S, G569S, S631N, H632T and Q633T in the amino acid sequence of SEQ ID NO. 1.
10. The modified HSV gB protein of claim 7, wherein said modification comprises a modification for introducing a sugar chain at a position corresponding to each of the amino acid residues D199, R567 and S631 in the amino acid sequence represented by SEQ ID NO. 1.
11. The modified HSV gB protein according to any one of claims 6 to 10, wherein said modification comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residue R602 in the amino acid sequence of SEQ ID NO. 1.
12. The modified HSV gB protein according to claim 11, wherein said sugar chain has been introduced by replacing the amino acid residues R602N, D603A and A604T in the amino acid sequence of SEQ ID NO. 1.
13. The modified HSV gB protein according to any one of claims 5 to 12, wherein said modification further comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residue D199 in the amino acid sequence of SEQ ID NO. 1.
14. The modified HSV gB protein according to claim 13, wherein said sugar chain has been introduced by replacing the amino acid residues D199N, D200A and H201T in the amino acid sequence represented by SEQ ID NO. 1.
15. The modified HSV gB protein of any one of claims 4 to 14, wherein the modification further comprises the substitution of the amino acid residue corresponding to arginine (R613) at position 613 in the amino acid sequence of SEQ ID NO. 1, to an alanine residue.
16. An HSV vaccine comprising the modified HSV gB protein of any one of claims 1-15.
17. A modified HSV gB protein which is a modified protein of the envelope glycoprotein B (gB) of Herpes Simplex Virus (HSV), wherein at least 1 amino acid residue present in a region which is located at a distance of 1.5nm or less from the amino acid residue corresponding to the 567 th arginine residue (R567), the 602 th arginine residue (R602), the 631 th serine residue (S631), or the 199 th aspartic acid residue (D199) in the amino acid sequence described in SEQ ID NO. 1 on the surface of the crystal structure of the extracellular domain of wild-type HSVgB is substituted or deleted.
18. The modified HSV gB protein of claim 17, wherein the modification comprises a modification for sugar chain introduction at a position of at least 1 amino acid residue selected from the group consisting of amino acid residues corresponding to D199, R567, R602, and S631 in the amino acid sequence of seq id No. 1.
19. The modified HSV gB protein of claim 17 or 18, wherein the modification comprises a modification for sugar chain introduction at a position corresponding to the amino acid residue D199 in the amino acid sequence of seq id No. 1.
20. The modified HSV gB protein of any one of claims 17 to 19, wherein the modification comprises a substitution of the amino acid residue corresponding to arginine (R613) at position 613 in the amino acid sequence of SEQ ID NO. 1, to an alanine residue.
21. The modified HSV gB protein according to any one of claims 17 to 20, wherein said modification comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residue R567 in the amino acid sequence of SEQ ID NO. 1.
22. The modified HSV gB protein of any one of claims 17 to 21, wherein said modification comprises a modification for introducing a sugar chain at a position corresponding to the amino acid residue S631 in the amino acid sequence of SEQ ID NO. 1.
23. An HSV vaccine comprising the modified HSV gB protein of any one of claims 18-22.
CN201880055586.8A 2017-08-30 2018-08-29 Modified HSV gB protein and HSV vaccine containing same Active CN111032681B (en)

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